Blood used in transfusion often causes post-transfusion infection. Hepatitis B virus (this may be hereinafter referred to as HBV) is a causative virus of post-transfusion hepatitis, which infects through transfusion in surgery or the like. Therefore, it is extremely important to diagnose whether or not the blood is infected with HBV by screening of the blood for transfusion. As a method to diagnose such HBV infection, a method to assay HBs antigen, in which HBs antigen is detected and quantified, is widely used.
HBs antigen is a major structural envelope protein on the surface of an infectious HBV particle and contained in the lipid bilayer membrane derived from hepatic cells, which membrane wraps the core particle containing HBV-DNA. Blood of a patient infected with HBV also contains noninfectious small spherical particles and tubular particles which are without the core particle and composed of HBs antigen. The small spherical particles exist in the blood most abundantly and are observed at ratios of about 1000 small spherical particles relative to 1 to several HBV particles. Currently available diagnostic reagents for HBs antigen detect mainly HBs antigen in the form of the small spherical particle.
HBs antigen is a membrane protein having the total length of 226 amino acid residues, which penetrates the lipid bilayer membrane 4 times. Although the model for the transmembrane structure of HBs antigen has not been completely elucidated yet, Howard et al. proposed that HBs antigen is made of: the first outer region (ER lumen side) of the lipid bilayer membrane, consisting of 1st to 11th residues of the N-terminus of HBs antigen; the first transmembrane region consisting of 12th to 28th amino acid residues, which penetrates the lipid bilayer membrane and is hydrophobic; the first inner region of the lipid bilayer membrane, consisting of 29th to 80th residues; the second transmembrane region consisting of 81st to 97th amino acid residues, which is hydrophobic; the second outer (ER lumen) region consisting of 98th to 156th amino acid residues, which is hydrophilic; and the third transmembrane region, second inner region, fourth transmembrane region and third outer (ER lumen) region consisting of the 157th amino acid residue and residues more distant from the N-terminus (Non-patent Literature 1: FIG. 1).
As the conventional method for assaying HBs antigen, methods using an antibody which binds to the common antigenic determinant a of HBs antigen have been common. The common antigenic determinant a of HBs antigen is located on the peptide consisting of 110th to 156th amino acid residues in the second outer (ER lumen) region of HBs antigen, that is, 98th to 156th amino acid residues. The common antigenic determinant a has a complex high dimensional structure and, furthermore, it is reported to have four epitopes (Non-patent Literature 2).
On the other hand, the above method for assaying HBs antigen using an antibody which binds to the common antigenic determinant a is sometimes incapable of detecting HBV having an amino acid mutation(s) in the region of HBs antigen. Therefore, a method for assaying HBs antigen using an antibody which binds to the peptide in the first inner region of the lipid bilayer was recently developed, which peptide is consisting of 26th to 80th amino acid residues (Patent Literature 1).
Patent Literature 1 WO 2006/033368
Non-Patent Literature 1: Howard et al. “Viral Hepatitis and Liver Disease” (Zuckerman A J and Alan R (eds.)) (Liss Inc, New York) 1988, pp. 1094-1101
Non-Patent Literature 2: Hiroaki Okamoto “Nippon Rinsho, Molecular Hepatitis Virology, Fundamental-Clinic-Prophylaxis, Lower Volume, Hepatitis A, B, D, E Viruses”, published on Oct. 26, 1995, pp. 212-222)